The Microscope For Microbiology Students

I. Microscope Use: Safety Rules 1. When transporting the microscope, hold it in an upright position with one hand on its arm & the other supporting its base. Avoid jarring the instrument when setting it down. 2. Use only special grit-free lens paper to clean the lenses. Clean all lenses before & after use. 3. Always begin the focusing process with the low power objective lens in position, changing to the higher-power lenses as necessary. 4. When using the microscopes, try to keep both eyes open to avoid getting a headache. 5. Use the coarse adjustment knob only with the low power objective lens. 6. Before leaving lab, remove any microscope slides from the stage, clean the oil immersion lens with lens paper, rotate the lowest-power objective lens into position, & cover the microscope. 7. Never remove any parts from the microscope; inform your instructor of any mechanical problems that arise. 8. Never yank on the electric cord of the microscope. Always pull it out of the plug by the base of the cord.

II. The Parts of the Microscope Utilizing the microscopes in lab and the diagram in your lab manual, identify the following microscope parts.  Arm  Base  Lamp  Rheostat - A device that uses resistance to regulate electrical current. The rheostat acts as a dimmer switch for the microscope. Please set this dial to 7 if present.  Focus Adjustment Knobs, Coarse and Fine  Condenser - a lens that serves to concentrate light from the lamp that is in turn focused through the specimen and magnified by one of the objective lenses. The condenser optimizes brightness, evenness of illumination, and contrast.  Substage (revolving) diaphragm  Stage with stage clips  Objective lenses - 4x (scanning), 10x (low power), 40x (high power), 100x (oil immersion)  Revolving nosepiece  Body tube (or optical head)  Ocular (eyepiece)

III. Magnification and Resolution

In a compound microscope, magnification is achieved through the interplay of two lenses, the ______lens and the ______lens.

The ______of the specimen is equal to the power of the ocular lens multiplied by the power of the objective lens used.

Why don't most microscopes magnify more than 1,000x? Although the level of magnification in a compound microscope is almost limitless, resolution is not. The ______(or resolving power) is the ability to distinguish that two objects, which are very close together, are, in fact, two separate objects. Remember what happens when you enlarge a blurry photo, the mistake just gets larger. What affects resolution? The amount & physical properties of the visible light that enters the microscope. In general, as light increases resolution ______.

IV. Viewing a prepared slides – the letter e and Crossed Threads Follow the procedure discussed in your lab manual and answer the questions on your data sheet.

 When using microscopes, after you have focused on the slide, you can change objectives and even change slides and only need to use the fine focus knob to sharpen the image because our microscopes are parfocalized.  The actual area of the slide that you can see through the ocular is called the ______. o As you increase magnification, you ______the field of view.  The vertical distance that is in focus in the slide is the ______. o As you increase magnification, you ______the depth of field

Microscopic Examination of Eukaryotic Microbes

Prokaryotes Eukaryotes Plants, Animals, Bacteria & Archaea Fungi, & Protists No membrane- Membrane-bound bound organelles organelles

70S ribosomes 80S ribosomes

Flagella Rotary Flagella whiplike movement movement DNA is single, DNA is 2+ linear circular chromosomes chromosome Reproduction Reproduction through binary through mitosis fission Eukaryotic Supergroups: Excavata, Archaeplastida, Chromalveolata, & Unikonta

Supergroup Excavata – usually have a feeding groove excavated from one side of the cell & have 1+ flagella. Many have an active feeding stage, the trophozoite, and an inert resting stage, the cyst.

Trichomonas vaginalis is the causative agent of vulvovaginitis (trichomoniasis) in human females. It has 4 anterior flagella and an undulating membrane made from a fin-like extension of the cytoplasmic membrane.

Giardia is the causative agent of giardiasis, a diarrheal disease commonly picked up by drinking untreated water that has been contaminated by animal droppings. Giardia have two large nuclei.

Euglena in action! http://youtu.be/0rNI8Bos_BQ

Euglena are unicellular protozoans that almost always have chloroplasts. Although they photosynthesize (autotrophy), Euglena can also eat food by heterotrophy (like animals) when light is unavailable. This makes them mixotrophic. They use 2 flagella for locomotion and have a distinctive red “eyespot.”

Supergroup Archaeplastida – members of this group descended from an ancestor that engulfed a cyanobacterium (by primary endosymbiosis), which ultimately evolved into the chloroplasts of green algae, red algae, and true plants. All members are autotrophic (they photosynthesize). Most also possess cell walls made of cellulose.

Volvox is a type of chlorophyte (known Watch this video of volvox in action! http://youtu.be/He9FSeGRi3A as green algae). It forms

spherical colonies of up

to 50,000 cells. They live

in a variety of freshwater

habitats. Each mature

Volvox colony is

composed of numerous

flagellate cells embedded in the surface of a hollow sphere. The cells swim in a coordinated fashion, with distinct anterior and posterior poles. The cells have They can also undergo sexual reproduction; eyespots which enable the colony to swim there are male colonies that produce sperm towards light. bundles and female colonies that produce eggs. The spheres will break up with advanced age. The generative cells then grow into new colonies; a type of asexual reproduction. Spirogyra are charophytes, which are even

more closely related to true plants. Spirogyra is a

filamentous green algae, named for the helical or

spiral arrangement of the chloroplasts. It is

commonly found in freshwater areas. Spirogyra

measures approximately 10 to 100μm in width and

may stretch centimeters long. This particular algal

species, commonly found in polluted water, is

often referred to as "pond scum". The cell wall

has two layers: the outer wall is composed of

pectin that dissolves in water to make the filament slimy to touch while the inner wall is of cellulose. The cytoplasm forms a thin lining between the cell wall and the large vacuole it surrounds. The chloroplasts are ribbon shaped, serrated or scalloped, and spirally arranged. In spring Spirogyra grows under water, but when there is enough sunlight and warmth they produce large amounts of oxygen, adhering as bubbles between the tangled filaments. The filamentous masses come to the surface and become visible as slimy green mats. Spirogyra cells are haploid. They undergo sexual reproduction when two filaments conjugate. The cytoplasm of two cells join, forming a diploid zygospore. When conditions are favorable, the zygospores germinate by undergoing meiosis, producing a new vegetative filament Supergroup Chromalveolata – members of this group descended from an ancestor that engulfed a red algal cell that evolved into a plastid with red algal characteristics. Because the red alga already had a chloroplast from primary endosymbiosis, the plastids in this group are said to have been formed by secondary endosymbiosis. In some modern species, the plastid is still present; in others, only red algal plastid DNA remains in the genome. Parmecium are unicellular, ciliated protozoans (microscopic animals). Simple cilia cover the body, which allow the cell to move with a synchronous motion (like a caterpillar) at speeds of approximately 12 body lengths per second. There is also a deep oral groove containing inconspicuous tongue-like compound oral cilia used to draw food into the cytosome or “mouth.” They generally feed on bacteria and other small cells. Osmoregulation is carried out by a pair of contractile vacuoles, which actively expel water from the cell absorbed by osmosis from their surroundings. Paramecia are widespread in freshwater environments.

Watch this video of paramecium in action! http://youtu.be/l9ymaSzcsdY

Plasmodium is a genus of parasitic protists. Humans can become infected with at least 11 species of Plasmodium, resulting in malaria. Malaria translates literally as “bad air.” It was supposed to be due to swam exhalations, but we now know it to be caused by a parasitic protozoan transmitted to humans by Anopheles mosquitoes. The protozoan occupies human red blood corpuscles (RBC’s), replicates inside them, and then destroys them as they escape to infect new RBC’s. This lifecycle results in intermittent or remittent fever characterized by attacks of chills, fever, and sweating.

Diatoms are a major group of golden brown algae, and are one of the most common types of phytoplankton (microscopic plants). Diatoms are primary producers within the food chain. Most diatoms are unicellular, although they can exist as colonies in the shape of filaments or ribbons, fans, zigzags, or stellate colonies. Diatom cells are characteristically encased within a unique cell wall made of silica (hydrated silicon dioxide) called a frustule. These frustules show a wide diversity in form, but usually consist of two symmetrical sides with a split between them, hence the group name. Supergroup Unikonta – this group is composed of heterotrophs and includes amoebas (of all sorts), animals, fungi, and others.

Amoebas are unicellular heterotrophs (they must eat food, as all other animals) that do not have cell walls. They obtain food through phagocytosis, utilizing cytoplasmic extensions called pseudopods to entrap their meals. Amoebas’ most recognizable features include one or more nuclei and a simple contractile vacuole to maintain osmotic equilibrium. There are parasitic amoebas.

Watch this video of an amoeba in action! http://youtu.be/W6rnhiMxtKU

Fungi are nonmotile eukaryotes. Their cell wall is usually made of chitin, not cellulose as in plants. Fungi are absorptive heterotrophs that secrete exoenzymes into their environment and then absorb the digested nutrients via hyphae. Abundant worldwide, fungi perform an essential role in the decomposition of organic matter and a fundamental role in nutrient cycling. Fungi have long been used as a source of food (mushrooms and truffles), as leavening in bread (yeast), and in fermenting food products (such as wine, beer, and soy sauce). Antibiotics, such as penicillin, have been produced from fungi. Most fungi are inconspicuous because of their small size and their cryptic lifestyle (living in the soil, feeding on dead matter). They become noticeable when fruiting, either as mushrooms or molds. Fungi may be saprophytes (getting their energy from decomposing nonliving organic matter), parasites (living at the expense of a living host organism), or symbionts (organisms that coexist with other organisms in a mutually beneficial relationship). Fungi are divided into unicellular yeasts and filamentous molds.

The Ascomycota, commonly known as the sac fungi, are the largest phylum of fungi. They produce an ascus (Greek “sac” or “wineskin”). The sac is a microscopic structure in which nonmotile spores (ascospores) are formed. This group is of particular importance to humans as they are sources of antibiotics (Penicillium), brewer’s yeast, baker’s yeast, and truffles. The fungal symbionts in the majority of lichens belong to Ascomycota. Saccharomyces cerevisiae which is used in the production of bread, wine, and beer, but is not an important human pathogen. Candida albicans is part of the normal respiratory, gastrointestinal, and female urogenital tract microbiota. Under the proper circumstances it may flourish and produce pathological conditions such as thrush in the oral cavity, vulvovaginitis of the female genitals, and cutaneous candidiasis of the skin.

Supergroup Unikonta cotinued– this group is composed of heterotrophs and includes amoebas (of all sorts), animals, fungi, and others.

Rhizopus are fast growing molds that produce white or grayish cottony mycelia. Rhizopus commonly causes rot in plants. Rhizopus grows as filamentous, branching hyphae that generally lack cross-walls. They can reproduce by forming asexual sporangiospores. In sexual reproduction, a dark zygospore is produced at the point where 2 compatible mycelia fuse. Nuclei between the 2 hyphae fuse, produce one or more diploid zygotes. After a dormant period, meiosis occurs. The zygospore then germinates and produces a sporangium similar to asexual sporangia. Haploid spores are then released to complete the lifecycle.